Histoplasma capsulatum is one of the classic dimorphic fungal pathogens, undergoing a temperature-induced transition from a mold form that grows in soil to a yeast form that establishes infection in lung macrophages. All of the dimorphic fungi cause the most severe disease in immunocompromised patients, but they are primary pathogens that can cause serious problems in hosts lacking any demonstrable immune defect. The first genetically proven virulence factor of H. capsulatum was CBP, a yeast phase-specific secreted protein that is important for proliferation within macrophages in vitro and in vivo. Solving the three-dimensional structure of CBP revealed a surprising similarity to saposin B, a mammalian protein involved in membrane processing and antigen presentation. Although the structural homology is strongly suggestive that CBP is the first identified fungal member of the large and diverse saposin-like protein family, its precise mechanistic role in histoplasmosis remains unproven. This grant application is designed to evaluate whether CBP indeed functions as a saposin and to understand the basis for CBP1 phase-specific regulation: Specific Aim 1. Define the saposin-like function of CBP. The studies in this Aim will first test whether CBP functions like a saposin in terms of binding lipids, either fungal or mammalian, and will include a structure-function analysis to determine which residues of CBP are involved. The potential role of CBP in CD1 antigen presentation, analogous to the function of saposin B, will also be explored. Specific Aim 2. Identify the genes involved in CBP1 transcriptional regulation. Insertional mutagenesis will be used to generate a library of mutants that will be screened for those that no longer regulate CBP1 normally. The most promising candidate regulatory genes will subsequently be used to identify coordinately regulated genes, potentially unraveling a network of genes related to CBP function or to other roles in pathogenesis. PUBLIC HEALTH RELEVANCE: Histoplasmosis is a common fungal respiratory infection that is endemic in the midwestern U.S., where the causative agent (Histoplasma capsulatum) grows in the soil and is responsible for hundreds of thousands of new infections annually. This research plan is designed to probe the mechanisms by which these organisms proliferate inside host cells and affect the host immune response. Because the primary focus is on a virulence factor that is uniquely associated with a pathogenic fungus, the results have the potential to guide development of novel antifungal drugs.